Augmentative intrinsic factor and process for preparing it in highly purified form



United States In. F; 4.-

AUGMENTATIVE INTRINSIC FACTOR AND PROC- ESS F OR PREPARING IT IN HIGHLYPURIFIED FORM No Drawing. Application January 17, 1956 Serial No.559,547

13 Claims. (Cl. 16774) This invention relates to improved methods ofpreparation of a therapeutic product having remarkable antianemiaefiicacy for treatable anemias.

More specifically, this invention relates to improved methods ofpreparation of intrinsic factor.

This invention also relates to an augmentative intrinsic factor andmethods of preparation of such augmentative intrinsic factor.

Intrinsic factor is a heat labile component of normal human gastricjuice which was first described by Castle and his associates, theAmerican Journal of Medical Science, vol. 178, page 748, in the year1929. Evidence presently indicates that intrinsic factor is involved inthe utilization of vitamin B In the classical condition in which adeficiency of intrinsic factor is found, namely pernicious anemia, smalloral doses of vitamin B are ineffective unless a source of intrinsicfactor is administered simultaneously. The study of gastric intestinalabsorption with radioactive vitamin B in pernicious anemia patients andhealthy individuals has proven that intrinsic factor is essential forthis absorption (Ellen bogen, Williams, Rabiner and LichtmanProceedingsof the Society for Experimental Biology and Medicine 89, 357, 1955).Although hematopoietic responses in pernicious anemia may follow theoral administration of massive doses of vitamin B given without theintrinsic factor, the absorption of the vitamin, when amounts comparableto those found in an average diet are ingested, involves a participationof intrinsic factor.

A number of methods of preparation of intrinsic factor have beendisclosed in the prior art; however, in all of them, difi'iculties havebeen encountered in that extremely low yields of therapeutically activeconcentrate of intrinsic factor are obtained as compared to the presentprocess. Therefore, the availability of sufficient quantities of suchtherapeutically active materials has been limited and the cost thereofhas been maintained at much higher levels than would be desired.

One of the principal objects of the present invention is, therefore, toprovide improved methods of preparing a therapeutically activeconcentrate of intrinsic factor from animal intestine for use intherapeutic compositions, and more particularly, it is to provideimproved and more efficient methods of preparing greatly increasedyields of substances having-intrinsic factor activity for use asdescribed above from animal duodenum, particularly hog duodenum.

It is a further principal object of the present invention to provideimproved methods of preparing a therapeutically active concentrate ofintrinsic factor from duodenum which possesses greater activity andpotency whereby the quantities of required daily dosage are reduced. 4

In a recently filed application, Serial No. 459,506, filed September 30,1954, and now abandoned, by one of the present inventors, a new methodis described for the preparation of intrinsic factor in considerablyhigher yields and of higher potency than that of the prior art. Thepresent atent G tunnel and kept frozen in a chilled room at 10 methodsare an improvement over ucts described therein.

The yield of the product produced by ammonium sulfate precipitation inthe instant process was about 15 times that of the above process(application Serial No. 459,506). Although being of the same potency, 30milligrams equaling one daily dose, the intrinsic factor concentratefrom the new process has a unique characteristic. The old processintrinsic factor (application Serial No. 459,506) as well as theproducts from other prior art processes inhibit the uptake of vitamin Bby healthy individuals whereas the preparation of intrinsic factor fromour new process in marked contrast unexpectedly increases the uptake ofthe vitamin B in healthy individuals. Of course, all intrinsic factorpreparations increase the uptake of vitamin B in pernicious anemiapatients, our product being unique only in its effect on persons who donot suffer from pernicious anemia. Obviously, this unique characteristicof the product produced by the present invention is very important.Vitamin B has long been known for its growth promoting activity. Sincethe new intrinsic factor of the present invention increases the uptakeof vitamin B it can be advantageously used, for not only the treatmentof anemias, but as a growth promoting factor. It will thus enable thesubject to absorb more of the vitamin B administered. As a consequence,it will make such therapy less expensive and more effectivelyadministered.

It is therefore a further principal object of the present invention toprovide a therapeutically active concentrate of intrinsic factorpossessing greater activity and potency that is augmentative to theuptake of vitamin B The present invention unexpectantly achieves thishighly desirable result.

In the present methods, the intrinsic factor concentrate is preparedfrom the small intestine of a commercially available animal, preferablythe duodenum, most desirably, the hog duodenum. The outside of thetissue is washed for a few seconds in water at about 30 C.; the insideof the tissue is not washed. Loose adhering fat is removed from theoutside of the tissue and the contents of the intestine, if present, areexpelled manually. If the intestine is not processed for intrinsicfactor immediately, the tissue is quickly frozen in flat layers with theaid of crushed Dry Ice or by the use of a wind C.

the methods and produntil used.

The intestine is then preferably fed into a meat grinder; it may,however, be minced with an axe or knife. The ground or minced tissue isthen slurried in a 0.5 to 3% solution of sodium chloride (preferably 2%)in the ratio of approximately one part of tissue by weight to 3 to 5parts of solution by volume. The intestine tissue is then slurried inthe saline solution and subsequently allowed to settle. A period of 30to 60 minutes has been found to be generally satisfactory for each ofthe slurrying step and the settling step. However, this time limitationis not generally critical; it will vary with the size of the batch andconcentration of the saline. As a matter of fact, as is illustratedbelow, the settling step is sometimes dispensed with entirely. Thetissue solids are then partially removed by filtration, decanting, orsome other separatory procedure. The solid residue is then preferablyextracted again once or several times with the saline solution, thesolids again partially removed, and the several extracts combined.

The pH of the combined extracts is then adjusted to a pH of from 7 to 10by the slow addition of an alkaline material such as sodium hydroxidewith stirring (a pH of 9 is preferred). This solution is allowed tostand to destroy pepsin. The pH is then adjusted to approximately 1.0 to2.5 and the solution allowed to stand to precipi- Patented Aug. 19, 1958tate out undesired muco-proteins pH of 1.5 to 2.0 is preferred).

and other impurities (a Thirty minutes has been generally found to be asatisfactory standing period for each of ithe pepsin'destroyingstep andthe muco-proteins destroying step, but for the reasonsset out above,this specific time limitation is not critical.

-The combined extracts obtained by the procedures described in thepreceding-paragraph are clarified by centrifugmg or some equivalentprocedure. Any standard centrifuge maybe used however, it is preferredto use the Sharples-'centrifuge model 'No. M'86PIE, maximum R. P. M.,15,000. ThepH of the clear filtrate is then adjusted to from about 3.9to 6.1, preferably 4.5 (the -'.ferred material; however, various commonsoluble inor- -ganic salts willperform this function. Moreover,varisodium sulfate; sodium chloride, etc. latter two categoriesarezincacetate plus ethanol and isoelectric point of intrinsic factoris-found in this range). A'material is thenadded to precipitate theintrinsic factor. -Ammoniumsulfate has heen' found to be the preousinorganic-organic salts or inorganic salts plus an alcohol are-alsosuitable- Examples of the first category are Examples of theferricchloride plus ethanol, respectively. Other equivalent proceduresfor precipitating proteinaceous materials are-found in the literature.See, for example, Amino- Acids-and Proteins-Therapy, Methods,Applications by David M.--Greenberg, published in 1951 by Charles C.Thomas; Springfield, Illinoikpa'ges 276 and 277 appear to be ofparticular interest. See also, The Proteins- Chemistry, BiologicalActivity and Methods, vol. I, part A, edited by Hans Neurath and KennethBailey, published in 1953 by Academic Press, Inc., New York, N. Y.,pages 49-to 57.

The solution-is allowed to stand until the precipitate is completelyformed. This standing period will generallyrun from /2 hour to 20 hours;16 hours has generally been found to be the optimum. This timelimitation is not critical as one can visually determine whenprecipitation is substantially complete. The precipitate is thencollected by centrifuging or some other separatory means.The'precipitate obtained in this manner may then be dialyzed to further.purify' the product. However, the

-' product obtained upon withoutdialysis. After dialysis, the materialmay be centrifuging is-suitable for use F freeze-dried or 'dried by-someother conventional method.

It has been indicated above that the various separatory procedures arenot critical in their identity. In other words, various proceduresequivalent to those described "-above' may be used. The separatoryprocedure for removing the tissue solids from the saline extract is,howi ever, one of the unique features of the present process. It is alsoone of the critical features of the present process in that through itsuse we have been able to obtain a product of much higher purity and muchhigher potency in much larger yields than anything heretofore produced.In the past, it 'hasbeen the practice to remove all solids from thesaline'extract; see Prusoff et al., The Journal of'l-lematology, vol.VIII, No. 6, pages 491 to 501 (particularly page 492), June 1953. Wehave unexpectedly i found that ifcontrolledamounts of the total solids(i. e.

tissue solids dissolved in the saline solution plus tissue solids whichare insoluble therein) are carried from the initial saline slurry phasethrough the separatory means a to the saline extract phase, the highyields of highly purified, highly active intrinsic factor are produced.

The amount of total tissue solids carried over into the "saline extractphase should be in the range of from about 35 %'to" about 60%-by weightof the initial weight of the tissue starting material. Both limits ofthis range are essential to the 'efficient operation of the instantprocess; the 'lower limit is critical, however, whereas the upper one isnot. If the total'tissue solids allowed to pass from the' slurry phaseto the-extract phase are substantially "lower-than 35%;the yield offinished product is very l substantially'reduced. i Ifthe' total'tissuesolids allowed centrifuge, as long as 4 to pass from the saline slurryphase to the saline extract phase substantially exceed 60%, then thepurity of the finished product may be materially reduced. However, bycarefully controlling conditions so that no undue contamination of theproduct results, it is plausible to exceed this upper limit of 60%. Forthe most part, this is not feasible, however, where large commerciallots of the product are being produced. A range of from'about 43% toabout 47% total tissue solids has been found to produce very goodresults, with a value of about45% total tissue solids producing optimumresults in terms of the yield, activity, and purity of the finishedproduct.

Filtering through cheesecloth is considered one of the best methods forseparating the solid tissue'from the aqueous saline solution in terms ofefiiciency, expense,

time and facility of operation. However, other methods may besubstituted therefor, the only limitation onthem being those describedabove with respect to the total solids allowed to pass from the'salineslurry'phase through the separatory means to the saline extract phase.As a matter of fact,*the Titan automatic intermittent sludge dischargecentrifuge (a' dish type machine having a feed rate of about 300 to 500gallons per hour'manufactured by the Pitmar Corporation of Baltimore,Maryland), is a better separatory device than cheesecloth iri'terms oftime and ease of operation. Other centrifuges, for example, theSuper-D-Canter, acontinuous' sludge discharge horizontal centrifuge madeby"Sharples'Corporation of Philadelphia, Pennsylvania, with the aid ofAlpha Flufi (Solka Floc product, comprising wood cellulose manufacturedby Brown Company, Boston, Massachusetts), may be used in place ofcheesecloth or the Titan centrifuge. Alpha Fluff is added to thetissueslurry at a concentration of'about 1%by slurry'we'ight. This addsstifiness to the meat particles thereby increasing the efliciency ofthe-Sup'er-D-Canter. Such equipmentusually operates at a feed rate of'about'300 to 400 gallons per hour. The Sharples centrifuge, model No.M-86 P-IE, referred to above has been tested and found to besatisfactory. Alternatively, a perforated basket type centrifuge may beused. Such a centrifuge has a liner associated with the basket. Acheesecloth liner has'been found to be satisfactory in this case. TheWestphalia hollow-bowl centrifuge made by Centrico of Englewood, NewJersey, using Alpha Fluff has also been tested and found to besatisfactory. Vacuum'filters such as the well-known Oliver filter may beused in'place of the cheesecloth or'the Titan centrifuge. Hydraulicpressing can be used in place of the cheesecloth or in addition tothecheesecloth or in conjunction with the "cheesecloth. Manualdecanting, of course, could be substituted for these various separatoryprocedures; other equivalent procedures will occur to those skilled inthe art.

The, exact composition of the saline solution'used throughout thisprocess is not critical. A" 2% saline solution has been indicated asbeing preferred; however, the limitations on-this concentration are onlythose of commercial practicality. The volume of saline solution canvary, but, of course, the greater the dilution the more costly theresulting voluminous solution is to handle. Similar considerations arisewith more'concentrated' solutions: i. e. precipitation of intrinsicfactor, cost of material, caking in the equipment used, etc.

It has been indicated that preferably the Sharples centrifuge referredto above be used toseparate the undesirable muco-protein solids andother impurities resulting from adjusting the pH to 1.5 to 2.0. It ispreferred since it most expeditiously removes the solids in terms oftime, expense and efficiency of operation. Other equivalent separatorymeans may be substituted for the Sharples they completely clarify thesolution. For example, the De Laval bowl typecontinuous desludgingcentrifuge model AOVO,-made by De Laval Separator Company, Poughkeepsie,New' York', has been used, and satisfactoryresults were obtained. Inaddition other separation equipment is suitable for this step, forexample, the well-known Nerofil filter aid (petroleum coke made by theDicalite Division of the Great Lakes Carbon Corporation) and ChrysoliteNo. 5 filter aid (a blend of asbestos and cellulose fibers made by theHercules Filter Company, Hawthorne, New Jersey). The above describedseparatory equipment may be used on Buchner funnels utilizing a vacuumto aid in separation.

The invention broadly described will be further illustrated in greaterdetail by the following specific examples.

It should be understood however, that although these examples maydescribe in particular detail some of the more specific features of thepresent invention, they are given primarily for the purpose ofillustration, and the invention in its broader aspects is not to beconstrued as limited thereto.

EXAMPLE 1 Process for the production of commercial intrinsic factorconcentrate starting with fresh unfrozen hog duodenum 1.75 kg. of hogduodenum from freshly killed hogs is fed into a meat grinder. The groundtissue is slurried in a 2.0% solution of NaCl in the ratio of 1 part ofduodenum by weight to 5 parts of solution by volume (8.75 liters of 2.0%NaCl). The saline is originally at a temperature of C. and is maintainedat this temperature in jacketed tanks containing water flowing throughthe jacket at this temperature. The ground tissue is stirred in the NaClsolution for 30 minutes, the agitation is stopped and the slurry formedthereby isailowed to settle for 30 minutes. After settling, the slurryis poured two times through one layer of cheesecloth. The residue isonce again extracted with one volume (1.75 liters) of 2% NaCl solutionand the two extracts are combined. The combined extracts are adjusted topH 9.0, by the slow addition of 5.0 N NaOH, with stirring. The solutionat this point is permitted to stand one-half hour. At the end of thistime, the solution is adjusted to pH 1.5 by the slow addition of 5.0 NHCl with stirring, and permitted again to stand for one-half hour. Atthe end of this time, a precipitate forms and it is removed by feedingit into a Sharples centrifuge (model #M86P-IE, maximum R. P. M. 15,000)at the rate of about 5.0 liters per minute at about 15,000 R. P. M. Theclear filtrate is then adjusted to pH 4.5 by the slow addition of 5.0 NNaOH with stirring. Ammonium sulfate is then added slowly with stirringot the filtrate in the amount of 368 gms. per liter of liquid. Thesolution is then left to stand 16 hours, at the end of which time theprecipitate is collected by Sharples centrifugation. The precipitate isremoved from the Sharples bowl and sufficient water is added to theprecipitate to obtain a thick slurry. This thick slurry is placed incellophane bags and is dialyzed for seven hours in a rotating laboratorydialyzer against cold running tap water. An air space is allowed in eachdialysis bag of such size that stirring is obtained inside the bagduring the dialysis. After dialysis the matter is freeze-dried.

EXAMPLE 2 Process for the production of commercial intrinsic factorconcentrate starting with frozen hog duodenum 10 kg. of frozen tissue ischopped with an ax in order to permit it to be fed while frozen into ameat grinder. The ground tissue is slurried in 30 liters of a 2.0%solution of NaCl (1 part of duodenum by weight to 3 parts of solution byvolume). The saline solution is originally at a temperature of 10 C.,and it is maintained at this temperature in a jacketed tank containingwater flowing through the jacket at about 10 C. The temperature of thesolution drops to about 3 to 5 C., upon the addition of frozen groundduodenum, but returns to 10 C. after about one-half hour. The groundduodenum is stirred in the NaCl solution for 30 minutes,

at the end of which time the agitation is stopped and the slurry formedthereby is allowed to settle. After 30 minutes of settling, the materialis poured through a double thickness of cheesecloth. The residue isextracted two times with one volume (10 liters) of 2.0% NaCl solutioneach time. The stirring and settling times are reduced to 10 minutes and15 minutes, respectively for later two re-extractions. The combinedextracts are then further processed exactly as described in Example 1except that 1.0 N NaOH and 1.0 N HCl are used instead of 5.0 N for pHadjustments, and the period of standing after the introduction of theprecipitating agent is extended to about 20 hours.

EXAMPLE 3 Preparation of highly purified intrinsic factor concentrateThe beginning of the process is essentially the same as described inExample 2 until after the centrifugation of the pH 1.5 ppt.

The supernatant from the pH 1.5 ppt. is adjusted to pH 4.5 by the slowaddition of 1.0 N NaOH, and ammonium sulfate is added to the supernatantin the amount of 174 gm./liter of liquid, slowly with stirring. Thesolution is then allowed to stand for four hours, at the end of whichtime it is centrifuged in the Sharples centrifuge at the rate of about0.5 liter per minute to obtain a clear filtrate. To the resultingfiltrate, 194 gm. of (NH SO per liter of filtrate is added slowly withstirring and allowed to stand for about 14 hours. This solution is thencentrifuged in the Sharples centrifuge at the rate of about 0.5 literper minute to obtain a clear filtrate. The precipitate is removed fromthe Sharples bowl and sufiicient water is added to obtain a thickslurry. The thick slurry is placed in cellophane membranes and dialyzedin a laboratory rotating dialyzer against cold running tap water forabout 4-6 hours. An air space is allowed in each bag of such size thatthorough stirring is obtained inside the bag. After dialysis thematerial inside the bag is centrifuged in the preparatory head of anultracentrifuge at 15,000 gravities for 15 minutes. The resultingprecipitate is washed with cold 2% NaCl solution and recentrifuged atthe same speed.

The residue is discarded and the supernatants are adjusted to pH 4.5with 1.0 N HCl. Ammonium sulfate is then added slowly to the supernatantin the amount of 368 gm./liter of solution and the solution is thenallowed to stand for about 14 hours. The resulting solution is thencentrifuged at 20,000 gravities for 10 minutes. The precipitate is onceagain suspended in water and dialyzed for 4-6 hours. After this dialysisthe material in the bag is centrifuged at 15,000 gravities for 15minutes and the precipitate is washed once with cold 2% NaCl solution.This precipitate is discarded and the clear solution is dialyzed for atleast 4 hours to free the sodium chloride. The material in the dialyzingbags is then freeze dried. This material is called reprecipitatedammonium sulfate fraction (RAS). It was tested at 5 mg. and found to beactive, as compared with the product from the production batch which wasactive at 30 mg. This fraction contains 2 to 3 peaks as observed byultracentrifugal analysis. The highest molecular weight peak(sedimentation constant of 4.0) seems to contain most of the activity ofthe highly purified RAS fraction and is separated from the low molecularweight material by ultracentrifugation as follows:

1.3 gm. of RAS fraction is dissolved in cc. of phosphate-saline bufferof pH 6.4 and subjected to a centrifugation of 100,000 gravities for 18hours. The resulting supernatant is collected; this contains the lowmolecular weight fraction. The pellet (or solid matter collected) isresuspended in phosphate-saline solution and centrifuged at 9,000gravities for 10 minutes. The resulting supernatant solution containsthe high molecular weight material. It has improved homogeneity and 7 itappears to contain rriost of the intrinsic factor activity (895 mg. oflow molecular weight material versus 321 mg. of highmolecular weightmaterial). This high molecular weight material has activity at about 2mg. (activity rated by adrninistration to pernicious anemia patients inrelapse).

EXAMPLE 4 The" process for theproiiu'ciion of t'bmmercial intrinsicfactor concentrate? in bill/C dinbzl'nts starting with' frozen hogduodenum 454 kg. of frozen tissue is chopped with an axe in order topermit it to be fed while frozen into a meat grinder. The ground tissueis slurried in 1,362 liters of a 2.0% solution of NaCl (1 part ofduodenum by weight to 3 parts of solution by volume). The salinesolution is originally at a temperature of C; and it is maintained atthis temperature in a jacketed tank containing water flowing through thejacket at about 10 C. The temperature'of thesolution drops to about 3 to5 C. upon the addition of frozen duodenum. The duodenum is stirred inthe NaCl solution for about50 minutes to form a slurry. The slurry ispumped while continuing to agitate to a No. 4 mesh screen which removeslong fibers. The 'slurryis' thenpumpe'd to a'Titan AutomaticIntermittent SludgeDis'char'ge Centrifuge to which it is introduced at arate of about 500 gallons per hour. The supernatant is collected as itis discharged. The sludge is collected as discharged "and thenre-extracted in 681 liters of the 2% saline solution (1 part of sludgeby weight to about 1 /2. parts'of saline solution by volume).

The re-extracted sludge'i's then stirred in the saline solution forabout 30 minutes atthe end of which time it is pumped again to'the'No. 4mesh screen while continuing to agitate and then tothe' Titan centrifugewhere the supernatant again is removed. The pH of the supernatants isthen adjusted to about 9.0 by the slow addition of 5.0 N NaOH withstirring. The solution at this point is then permitted to stand V2 hour.At the end of this time the solution is adjusted to a pH of about 2.0 bythe slow addition of 50% HQ with stirring and permitted again to standfor /2 hour. At the end of this time a precipitate forms and'itis'removed by feeding it into a Sharples centrifuge (model No. M-86P-IE,maximum R. P. M. 15,000) at the rate of about 0.5

liter per minute at about 15,000 R. P. M. The clear filtrate is thenadjusted to a 'pH of about 4.5 by the slow addition of a 5.0 N NaOH withstirring. Ammonium sulfate is then added slowly with stirring to thefiltrate in the amount of 368 grams per liter of liquid, the solution isthen left to stand for about 16 hours, at the end of which time theprecipitate is collected by a Sharples centrifugation. The precipitateis removed from the Sharples bowl and sufiicient water is added to theprecipitnte to obtain a thick paste. This thick slurry is placed inviscose-cellulose tubing and is dialyzed for about 27 hours in anoscillating dialyzer against cold running tap water. An air space isallowed in each dialysis tube of such size that stirring is obtainedinside the tube during the dialysis. After dialysis, the material isfreeze-dried.

EXAMPLE 5 The procedure of Example 4 is repeated utilizing all of thesame quantities, temperatures, etc. However, the Westphalia hollow-bowlcentrifuge is used in place of the Sharples' centrifuge after both the2.0 pH adjustment and in collecting the ammonium sulfate precipitate ata feed rate of 300 gallons per hour. Moreover, the feed rate into theTitan centrifuge is 300 gallons per hour; the solution is left tostandtwelve hours after adding the ammonium sulfate to it, and theresultingprecipitate is dialyzed for 24 hours.

8 EXAMPLE 6 The procedure of Example 4 is again repeated, however, inthis case the Titan automatic intermittent sludge discharge centrifugeis substituted for the Sharples centrifuge after the 2.0 pH adjustmentstep. Moreover, the solution is left to stand two hours after theammonium sulfate is added; the precipitate is dialyzed for 30 hours, andthe feed rate of the Titan centrifuge is 400' gallons per hour.

The following absorption experiments illustrate the unique andunexpected property of the product produced by the instant process; i.e. its augmentative quality in healthy individuals:

GENERAL TECHNIQUE Each treatment was made on a group consisting of tenyOung healthy males. All individuals received a g. oral dose ofradioactive vitamin B and 1 g. of unlabeled vitamin B parenterally.Their urine was collected for 24 hours. Diets were the same for all,both before and during the experiments. The amount of radioactivevitamin B excreted is a measure of the amount absorbed from the gut intothe tissues. The higher the urinary excretion the higher was thegastrointestinal absorption. In each of the following tables the variousamounts of intrinsic factor concentrates used are equivalent to 4 dailyoral doses as determined from pernicious anemia cases in relapse.

1ST EXAMPLE 1 Standard error of the mean. Efiect of various intrinsicfactor preparations on absorption of vitamin B by healthy subjects 2NDEXAMPLE Milliml- Intrinsic Factor Preparacrograms tion administeredVitamin B12 Comments Excreted In 24' Hours Group 1. None 490i31. 8Absorption due to person's own intrinsic factor.

Group 2. 100 mg.intri.nstcfactor From 364i25. 5 Inhibits Absorp- GompanyAs dried hog. tion. Stomach (application Serial No. 459,500).

Group 3. 100 mg. intrinsic factor (tna- 639. 5i63. 4 Augments furtherterial from fresh hog duoabsorption. denum prccesstl1is apv plication).

Group 4. 100 mg. intrinsic factor-.- 42831311 Inhibits Absorpprocess ofthis application.

tion using hog stomach.

cation Serial No. 459,506 inhibits the uptake of vitamin B from the gutof young healthy males who already possess intrinsic factor in their ownstomachs. As shown in the 1st Example, group 5, company As intrinsicfactor concentrate which is made from hog stomach is also inhibitory. Ofspecial importance are groups 3 and 4 of Example 2. The duodenums andstomachs were removed from the same 25 hogs and processed separately ina fresh tissue process on the same day. The intrinsic factor from theduodenums was used in group 3 and the stomachs in group 4. Therefore, itwas found unexpectedly that duodenum does not contain the material whichinhibits B uptake whereas the stomach does. This further illustrates theuniqueness of intrinsic factor produced from duodenum by the processdescribed in this application.

Effect of various intrinsic factor preparations an absorption of vitaminB by healthy subjects 3RD EXAMPLE Millimi- Intrinsic FactorPreparaerograms tion Administered Vitamin B12 Comments Excreted In 24Hours Group 1. None 528.6=l=37.8 Absorption due to persons own intrinsicfactor. Group 2 100 mg. intrinsic factor as 394.7=|:62.3 InhibitsAbsorpmade in application Setion. rial N 0. 459,506. Group 8- 200 mg.Company B's in- 492.6:b89.4 Do.

trinsic factor concentrate. Group 4. 800 mg. Company Us in- 497.4=l=50.1Do.

trinsic factor concentrate.

1 Standard error of the mean.

10 adjusting the pH to approximately 1.0 to 2.5 to precipitate outundesired muco-proteins and other impurities, removing the resultantprecipitate from the solution, discarding the thus removed precipitate,adjusting the pH of the clear filtrate to about 3.9 to 6.1, adding aprecipitating agent to precipitate the intrinsic factor.

2. The process of preparing augmentative intrinsic factor whichcomprises comminuting hog duodenum, slurrying said comminuted hogduodenum in a 2% saline solution, partially removing tissue solids toproduce an extract phase containing approximately 45% total tissuesolids, adjusting the pH of the extract phase to approximately 9 inorder to destroy pepsin, then adjusting the pH to approximately 1.5 to2.0 to precipitate out undesired muco-proteins and other impurities,removing the resultant precipitate from the solution and discarding it,adjusting the pH of the clear filtrate to approximately 4.5, addingammonium sulfate to precipitate the crude intrinsic factor, purifyingthis precipitate by dialysis followed by freeze-drying.

3. The process of claim 1 wherein the extract phase contains from about35% to about total tissue solids.

4. The process of claim 1 wherein the extract phase contains from about43% to 47% total tissue solids.

5. The process of claim 1 wherein the animal intestine material is hogduodenum.

6. The process of claim 1 wherein the saline solution is one containingfrom about 0.5 to 3% sodium chloride.

7. The process of claim 1 wherein the precipitating agent comprisesammonium sulfate.

8. The process of claim 1 wherein the precipitating agent comprisessodium sulfate.

9. The process of claim 1 wherein the precipitating agent comprisessodium chloride.

10. The process of claim 1 wherein the agent comprises zinc acetate plusethanol.

11. The process of claim 1 wherein the precipitating agent comprisesferric chloride plus ethanol.

12. The process of claim 1 wherein the intrinsic factor is furtherpurified 'by dialysis and then freeze-dried.

13. Augmentative intrinsic factor prepared by the method of claim 1.

precipitating References Cited in the file of this patent Prusofi:Blood, The J. of Hematology, vol. 9, No. 6, June 1953, PP. 491-501(part. pp. 492-494).

1. A PROCESS FOR PREPARING AUGMENTATIVE INTRINSIC FACTOR WHICH COMPRISESCOMMINUTING ANIMAL INTENSITINE MATERIAL, SLURRY SAID COMMINUTEDINTESTINE MATERIAL IN A SALINE SOLUTION, PARTIALLY REMOVING TISSUESOLIDS TO PRODUCE AN EXTRACT PHASE CONTAINING AT LEAST 35% TOTAL TISSUESOLIDS, ADJUSTING THE PH OF THE EXTRACT PHASE TO APPROXIMATELY 7 TO 10IN ORDER TO DESTROY PEPSIN, THEN ADJUSTING THE PH TO APPROXIMATELY 1.0TO 2.5 TO PRECIPITATE OUT UNDESIRED MUCO-PROTEINS AND OTHER IMPURITIES,REMOVING THE RESULTANT PRECIPITATE FROM THE SOLUTION, DISCARDING THETHUS REMOVED PRECIPITATED, ADJUSTING THE PH OF THE CLEAR FILTRATE TOABOUT 3.9 TO 6.1, ADDING A PRECIPITATING AGENT TO PRECIPITATE THEINTRINSIC FACTOR.